The presence of long chains of alpha2, 8-polysialic acid (PSA) on cell surface glycoproteins negatively modulates cell adhesion by preventing cells from closely apposing one another. During development, PSA functions in axon guidance, axon pathfinding and cell migration. In the adult animal, its expression persists in areas of the brain requiring morphofunctional plasticity, and it is re-expressed in various cancer cells where it is thought to contribute to their highly metastatic behavior. PSA is a protein-specific modification that is found on a small group of proteins that includes the neural cell adhesion molecule (NCAM), the a subunit of the voltage-dependent sodium channel, and the polysialyltransferases PST and STX. What directs the polysialyltransferases to recognize specific glycoproteins is not known. We hypothesize that the polysialyltransferases recognize some amino acid sequence or structural feature of the glycoprotein substrate, and that this initial protein-protein interaction allows the specific polysialylation of oligosaccharides. In the first aim of this proposal we will elucidate the protein signals mediating NCAM polysialylation. We have established that the two polysialyltransferases, PST and STX, are autopolysialylated in cells. In the second aim of this proposal we will investigate the process of enzyme autopolysialylation and how the autopolysialylation of PST impacts NCAM polysialylation. We demonstrated that the MCF7 human breast carcinoma line and the RBL rat basophilic leukemia line express high levels of PSA and PST, but not NCAM or the sodium channel. We hypothesize that the PSA detected in these cells is modifying endogenous PST. In the third specific aim, we will determine what polysialylated proteins are present in these cells to test the hypothesis that autopolysialylated PST is expressed at appropriate levels and locations to impact the interactions of these cells. The overall goal of these and future studies is to understand the mechanism of protein-specific polysialylation and the role that PSA plays in the function of the proteins it modifies.